1. Field of Invention
This invention relates generally to biosensors for measuring physiological analytes in humans, and particularly to biosensors suitable for implantation to provide in vivo monitoring of a selected analyte, such as monitoring of blood glucose levels in diabetics.
2. Description of Related Art
For some time the medical community has recognized a need for implantable biosensors to monitor physiologically important analytes. The need is particularly pressing for continuous monitoring of blood glucose in diabetics, since failure to properly maintain glucose levels leads to serious consequences in both the short and long term. The long-term consequences include kidney failure, blindness, and amputation. To date, however, the only test readily available is a fingerstick kit, which does not provide continuous monitoring. Most diabetics use such kits infrequently at best, because of the pain and inconvenience associated with them.
In developing various implantable devices, hydrogels have been widely used as protective biocompatible coatings for the devices. Hydrogels are generally defined as polymeric materials that swell in water and other fluids, absorbing the fluid within the polymer network without dissolving. Hydrophilic hydrogels have a large amount of water content at equilibrium, and good biocompatibility.
Brock et al. describe attempts to make artificial muscle from bundles of extremely thin (10 micron diameter) polyacrylonitrile fibers (xe2x80x9cDynamic Model of Linear Actuator based on Polymer Hydrogelxe2x80x9d, published on the Web at www.ai.mit.edu/projects/muscle/papers/icim94). The fiber bundles are attached to an artificial tendon with epoxy and thence to a mechanical linkage; the swelling and contraction of the fibers is manipulated by alternately irrigating with acidic and basic solutions. Hydrogels also can be used to mimic human tissue.
The above-described properties of hydrogels make them attractive for use in implantable biosensors. One such device is an implantable amperometric sensor intended to measure glucose levels in blood or body fluids (U.S. Pat. No. 4,703,756 to Gough et al.). A second type of hydrogel biosensor which uses a pressure transducer to measure changes in osmotic pressure in pH-sensitive hydrogels, developed by the present inventors, is described in U.S. Pat. No. 6,268,161 to Han et al., and in co-pending U.S. patent application Ser. Nos. 09/308,392 and 09/644,323.
The Gough et al. biosensor uses the enzymatic activity of glucose oxidase (GOX) to measure glucose levels. GOX catalyzes the conversion of glucose to gluconic acid and hydrogen peroxide (H2O2), consuming oxygen in the process. The GOX reaction can be followed using electrochemical transducers of various kinds, but the most advanced type of device is the amperometric sensor. In the amperometric method, an electrode produces a current proportional to the diffusional flux of hydrogen peroxide to the electrode surface, or, alternatively, proportional to the diffusional flux of oxygen (O2) to the electrode surface. An increase in the surrounding glucose concentration should increase the diffusional flux of glucose into the membrane and increase the reaction rate within the membrane. The increase in enzymatic reaction rate in turn should increase the local hydrogen peroxide concentration and decrease the local oxygen concentration within the membrane. This increases the current detected by a hydrogen peroxide-based electrode sensor, or decreases the current to an oxygen-based electrode sensor. The latter approach, based on detecting the oxygen flux, requires a reference oxygen-based electrode sensor located in a hydrogel without the enzyme.
A second class of osmotic-pressure hydrogel sensors uses a pressure transducer to directly measure osmotic pressure changes in a hydrogel disposed within a rigid chamber having one open side which is covered with a flexible, semi-permeable diaphragm (Han et al., U.S. Pat. No. 6,268,161; Han et al., U.S. application Ser. Nos. 09/839,993 and 09/644,323). The pressure transducer senses changes in the pressure exerted by the hydrogel on the flexible diaphragm. Two types of such sensors have been developed. One uses pH-sensitive hydrogels having immobilized GOX. In this device, the gluconic acid produced by enzymatic action of GOX on free glucose changes the pH in the fluid matrix, causing it to swell (if the hydrogel has pendant acidic groups) or to shrink (if the hydrogel has pendant basic groups). The second type, which has potentially far wider application, uses the principles of the competitive binding assay. Both analyte and analyte-binding molecules are immobilized within the hydrogel; noncovalent bonds between the two effectively produce crosslinks. When free analyte displaces immobilized analyte, the crosslinking index changes, producing either swelling or shrinking of the hydrogel (depending on other factors in hydrogel composition). The resulting changes in osmotic pressure are measured with a pressure transducer in the same way as for the GOX osmotic-pressure biosensor. Where the analyte is glucose, the immobilized analyte binding molecule may for example be concanavalin A.
A disadvantage of the first two implantable hydrogel-based biosensors described above is the need for a battery for operation of the sensor""s transducers and/or for telemetry of sensor readings. Since these are ideally implanted in the patient""s body for continuous monitoring of the analyte, repeated invasive procedures are required whenever the battery must be replaced.
Further, so far as we are aware, there is no hydrogel biosensor system in which swelling of an implanted hydrogel is monitored by image capture from outside the body. While ATS Laboratories, Inc. (404 Knowlton St., Bridgeport, Conn. 06608; or on the Web at atslabs.com) manufactures hydrogel phantoms for use in quality control testing of ultrasound machines, these phantoms are strictly external devices, not implanted. Thus, there remains a need for an implantable analyte-sensitive biosensor chip which can be monitored by external imaging.
A biosensor chip system comprises an analyte-sensitive hydrogel slab chemically configured to vary its displacement volume according to changes in concentration of an analyte in a patient""s body fluid, in combination with external scanning means disposed and configured to quantifiably detect changes in the displacement volume of the hydrogel slab. The slab is preferably disposed within an enclosure, channel, or groove on a support block made of rigid or semi-rigid support material; the groove has one or more openings covered with a semipermeable membrane to allow contact between the patient""s body fluid and the hydrogel. In a preferred embodiment, the groove is configured to permit expansion of the hydrogel in substantially only one dimension. In a highly preferred embodiment, the hydrogel slab has an elongated filament-like shape, with the length at least about 5 to 50 times the crosswise dimension(s). The scanning means may be any type of imaging device capable of resolving changes in the slab""s dimensions when it is implanted within a patient. In a highly presently preferred embodiment, the scanning means is a handheld ultrasound unit; however, other image scanning means including magnetic resonance imagers (MRI) and computerized tomographic scanners (CT) could be used.
Desirably, the biosensor chip also includes a second groove with a reference hydrogel slab, and/or scale marks which can be imaged together with the hydrogel slab to provide a precise dimensional calibration.
The analyte-sensitive slab may be made of any material that alters its displacement volume in response to a change in analyte concentration. Two types of specially chemically configured hydrogels are presently preferred for use in the invention. In one, an oxidative enzyme is immobilized within a pH-sensitive hydrogel, and catalyzes a reaction of the analyte to produce a charged product. The term xe2x80x98pH-sensitive hydrogelxe2x80x99 refers generally to a hydrogel modified to contain pendant charged groups in proportions that produce an overall acidic or basic environment in the fluid within the gel. The immobilized enzyme might be, for example, glucose oxidase, GOX, where the analyte to be measured is glucose. The charged product generated by activity of the enzyme on the analyte causes the hydrogel to change its displacement volume (swell or shrink), which changes can be detected by the scanning means. The second type of hydrogel has both analyte binding molecules (ABMs) and analyte or analyte analogue molecules (AAMs) co-immobilized within it, in addition to charged pendant groups. In the absence of free analyte, immobilized ABMs bind to immobilized AAMs, forming what are in effect non-covalent xe2x80x98crosslinksxe2x80x99. As free analyte from a body fluid or test solution diffuses into the hydrogel, binding competition displaces immobilized AAMs from ABMs, thus reducing the number of xe2x80x98crosslinksxe2x80x99. This reduction in crosslinking causes swelling of the hydrogel.
Also, in addition to the above two types of hydrogels, it is within contemplation that other analyte-sensitive swellable materials, polymers, and hydrogels meeting that description may be developed and will be useful in the biosensor. Certain embodiments of the invention are specifically designed to detect glucose levels in body fluids.
In its broadest conception, the invention is an implantable biosensor chip containing an analyte-sensitive hydrogel, which can be imaged by a non-invasive external scanning means such as ultrasound. Such a biosensor chip comprises a support block formed of rigid or semi-rigid, biocompatible material; an enclosure, channel, or groove in the support block having one or more openings permitting penetration of a patient""s body fluid; and an analyte-sensitive hydrogel slab disposed within the groove, the hydrogel being chemically configured to vary its displacement volume according to changes in concentration of an analyte in the body fluid.
The invention further encompasses methods of determining the concentration of free analyte in a solution and of making the biosensor chip. The method of determining analyte concentration comprises steps of: providing a hydrogel having pendant charged and/or uncharged moieties, analyte molecules, and analyte-specific binding molecules covalently immobilized therein; contacting the hydrogel sequentially with a series of calibration solutions having known concentrations of free analyte, and measuring the displacement change in the hydrogel for each of the calibration solutions to produce a calibration curve of displacement change versus analyte concentration; contacting the hydrogel with the test fluid, and measuring a resulting displacement change; and comparing the resulting displacement change with the calibration curve to determine the analyte concentration of the test fluid. A further embodiment of the method includes a step of enclosing the hydrogel in a rigid or semi-rigid structure which has at least one permeable portion through which free analyte in the test solution can diffuse into the hydrogel, the structure sized and configured to permit hydrogel expansion in substantially only one dimension.